The beneficial effect of cold atmospheric plasma on parameters of molecules and cell function involved in wound healing in human osteoblast-like cells in vitro.

The aim of this study was to analyse the effect of cold atmospheric plasma (CAP) on human osteoblast-like cells in vitro. Additionally, underlying intracellular mechanisms were to be studied. Human osteoblast-like (MG63) cells were exposed to CAP for 60 s. The effects of CAP on key molecules essential for the wound healing response were studied using real-time PCR, ELISA and immunocytochemistry. For studying intracellular signalling pathways, MAP kinase MEK 1/2 was blocked. Cell viability was analysed by an XTT assay and with an EVE automated cell counter. Cell migration was examined by an in vitro wound healing assay.CAP exposition on osteoblast-like cells caused a significant upregulation of interleukin (IL)-1ß, IL-6, IL-8, tumor necrosis factor (TNF)α, cyclooxygenase (COX)2, collagen (COL) 1α, matrix metalloproteinase (MMP)1, Ki67, proliferating-cell-nuclear-antigen (PCNA) and chemokine ligand (CCL)2 mRNA expression at 1 day. Interestingly, after blocking of MAP kinase, CAP-induced upregulation of Ki67 was inhibited by 57%. Moreover, CAP treatment improved significantly osteoblast-like cell viability as compared to untreated cells at 1 day. Beneficial effect of CAP treatment was shown by an in vitro wound healing assay, displaying a significant faster wound closure. Our findings provide evidence that CAP exposure effects gene and protein regulation in human osteoblast-like cells. Furthermore, CAP treatment has a positive impact on wound closure in an in vitro setting and might improve existing concepts of hard tissue regeneration in the future.

Regulation of the autophagy-marker Sequestosome 1 in periodontal cells and tissues by biomechanical loading.

PURPOSE: Orthodontic treatment is based on the principle of force application to teeth and subsequently to the surrounding tissues and periodontal cells. Sequestosome 1 (SQSTM1) is a well-known marker for autophagy, which is an important cellular mechanism of adaptation to stress. The aim of this study was to analyze whether biomechanical loading conditions regulate SQSTM1 in periodontal cells and tissues, thereby providing further information on the role of autophagy in orthodontic tooth movement. METHODS: Periodontal ligament (PDL) fibroblasts were exposed to cyclic tensile strain of low magnitude (3%, CTSL), and the regulation of autophagy-associated targets was determined with an array-based approach. SQSTM1 was selected for further biomechanical loading experiments with dynamic and static tensile strain and assessed via real-time polymerase chain reaction (RT-PCR) and immunoblotting. Signaling pathways involved in SQSTM1 activation were analyzed by using specific inhibitors, including an autophagy inhibitor. Finally, SQSTM1 expression was analyzed in gingival biopsies and histological sections of rats in presence and absence of orthodontic forces. RESULTS: Multiple autophagy-associated targets were regulated by CTSL in PDL fibroblasts. All biomechanical loading conditions tested increased the SQSTM1 expression significantly. Stimulatory effects of CTSL on SQSTM1 expression were diminished by inhibition of the c­Jun N­terminal kinase (JNK) pathway and of autophagy. Increased SQSTM1 levels after CTSL were confirmed by immunoblotting. Orthodontic force application also led to significantly elevated SQTSM1 levels in the gingiva and PDL of treated animals as compared to control. CONCLUSIONS: Our in vitro and in vivo findings provide evidence of a role of SQSTM1 and thereby autophagy in orthodontic tooth movement.

Role of Cathepsin S in Periodontal Inflammation and Infection.

Cathepsin S is a cysteine protease and regulator of autophagy with possible involvement in periodontitis. The objective of this study was to investigate whether cathepsin S is involved in the pathogenesis of periodontal diseases. Human periodontal fibroblasts were cultured under inflammatory and infectious conditions elicited by interleukin-1ß and Fusobacterium nucleatum, respectively. An array-based approach was used to analyze differential expression of autophagy-associated genes. Cathepsin S was upregulated most strongly and thus further studied in vitro at gene and protein levels. In vivo, gingival tissue biopsies from rats with ligature-induced periodontitis and from periodontitis patients were also analyzed at transcriptional and protein levels. Multiple gene expression changes due to interleukin-1ß and F. nucleatum were observed in vitro. Both stimulants caused a significant cathepsin S upregulation. A significantly elevated cathepsin S expression in gingival biopsies from rats with experimental periodontitis was found in vivo, as compared to that from control. Gingival biopsies from periodontitis patients showed a significantly higher cathepsin S expression than those from healthy gingiva. Our findings provide original evidence that cathepsin S is increased in periodontal cells and tissues under inflammatory and infectious conditions, suggesting a critical role of this autophagy-associated molecule in the pathogenesis of periodontitis.

Regulation of p53 under hypoxic and inflammatory conditions in periodontium.

OBJECTIVES: Different studies suggest that inflammation as well as hypoxia leads to an increase of p53 protein levels. However, the implication of p53 during oral inflammatory processes is still unknown. The aim of this study was therefore to investigate the effect of hypoxia and inflammation on p53 regulation in human periodontium in vitro and in vivo. MATERIALS AND METHODS: Under hypoxic and normoxic conditions, human primary periodontal ligament (PDL) fibroblasts (n = 9) were stimulated with lipopolysaccharides (LPS) from Porphyromonas gingivalis (P.g.), a periodontal pathogenic bacterium. After different time points, cell viability was tested; p53 gene expression, protein synthesis, and activation were measured using quantitative RT-PCR, immunoblotting, and immunofluorescence. Moreover, healthy and inflamed periodontal tissues were obtained from 12 donors to analyze p53 protein in oral inflammatory diseases by immunohistochemistry. RESULTS: LPS-P.g. and hypoxia initially induced a significant upregulation of p53 mRNA expression and p53 protein levels. Nuclear translocation of p53 after inflammatory stimulation supported these findings. Hypoxia first enhanced p53 levels, but after 24 h of incubation, protein levels decreased, which was accompanied by an improvement of PDL cell viability. Immunohistochemistry revealed an elevation of p53 immunoreactivity in accordance to the progression of periodontal inflammation. CONCLUSIONS: Our data indicate that p53 plays a pivotal role in PDL cell homeostasis and seems to be upregulated in oral inflammatory diseases. CLINICAL RELEVANCE: Upregulation of p53 may promote the destruction of periodontal integrity. A possible relationship with carcinogenesis may be discussed.

Hypoxia and P. gingivalis synergistically induce HIF-1 and NF-κB activation in PDL cells and periodontal diseases.

Periodontitis is characterized by deep periodontal pockets favoring the proliferation of anaerobic bacteria like Porphyromonas gingivalis (P. gingivalis), a periodontal pathogen frequently observed in patients suffering from periodontal inflammation. Therefore, the aim of the present study was to investigate the signaling pathways activated by lipopolysaccharide (LPS) of P. gingivalis (LPS-PG) and hypoxia in periodontal ligament (PDL) cells. The relevant transcription factors nuclear factor-kappa B (NF-κB) and hypoxia inducible factor-1 (HIF-1) were determined. In addition, we analyzed the expression of interleukin- (IL-) 1ß, matrix metalloproteinase-1 (MMP-1), and vascular endothelial growth factor (VEGF) in PDL cells on mRNA and protein level. This was accomplished by immunohistochemistry of healthy and inflamed periodontal tissues. We detected time-dependent additive effects of LPS-PG and hypoxia on NF-κB and HIF-1α activation in PDL cells followed by an upregulation of IL-1ß, MMP-1, and VEGF expression. Immunohistochemistry performed on tissue samples of gingivitis and periodontitis displayed an increase of NF-κB, HIF-1, and VEGF immunoreactivity in accordance with disease progression validating the importance of the in vitro results. To conclude, the present study underlines the significance of NF-κB and HIF-1α and their target genes VEGF, IL-1ß, and MMP-1 in P. gingivalis and hypoxia induced periodontal inflammatory processes.

LPS from P. gingivalis and hypoxia increases oxidative stress in periodontal ligament fibroblasts and contributes to periodontitis.

Oxidative stress is characterized by an accumulation of reactive oxygen species (ROS) and plays a key role in the progression of inflammatory diseases. We hypothesize that hypoxic and inflammatory events induce oxidative stress in the periodontal ligament (PDL) by activating NOX4. Human primary PDL fibroblasts were stimulated with lipopolysaccharide from Porphyromonas gingivalis (LPS-PG), a periodontal pathogen bacterium under normoxic and hypoxic conditions. By quantitative PCR, immunoblot, immunostaining, and a specific ROS assay we determined the amount of NOX4, ROS, and several redox systems. Healthy and inflamed periodontal tissues were collected to evaluate NOX4 and redox systems by immunohistochemistry. We found significantly increased NOX4 levels after hypoxic or inflammatory stimulation in PDL cells (P < 0.001) which was even more pronounced after combination of the stimuli. This was accompanied by a significant upregulation of ROS and catalase (P < 0.001). However, prolonged incubation with both stimuli induced a reduction of catalase indicating a collapse of the protective machinery favoring ROS increase and the progression of inflammatory oral diseases. Analysis of inflamed tissues confirmed our hypothesis. In conclusion, we demonstrated that the interplay of NOX4 and redox systems is crucial for ROS formation which plays a pivotal role during oral diseases.

Gene analysis of signal transduction factors and transcription factors in periodontal ligament cells following application of dynamic strain.

OBJECTIVE: Orthodontic treatment is usually associated with the application of forces to teeth and periodontium. Instrumental in transmitting these forces are the cells of the periodontal ligament (PDL). In the present study, we used an established strain model to investigate the potential role of biophysical stimulation in modulating the gene expression pattern of these PDL cells. MATERIALS AND METHODS: PDL cells derived from non-carious and periodontally healthy teeth of six patients were grown on culture plates coated with collagen type I. Upon completion of culture, dynamic strain was applied to the cells for 24 h, using 3% of tensile force and a frequency of 0.05 Hz. This loading protocol for biomechanical stimulation was followed by extracting the RNA from the cells and using a RT(2) PCR array(®) for analysis. RESULTS: Compared to non-stimulated control cells, this analysis revealed the induction of several factors (e.g., RELA, IRF1, MAX, MYC, CDKN1B, BCL2, BCL2A1) known to influence tissue homeostasis by contributing essentially to cell proliferation, cell differentiation, and the inhibition of apoptosis. CONCLUSION: This study demonstrates that the biomechanical stimulation of PDL cells is an important factor in periodontal tissue homeostasis.